Open Access
Open Peer Review

This article has Open Peer Review reports available.

How does Open Peer Review work?

Inhibitory activities of selected Sudanese medicinal plants on Porphyromonas gingivalis and matrix metalloproteinase-9 and isolation of bioactive compounds from Combretum hartmannianum (Schweinf) bark

  • Ebtihal Abdalla M. Mohieldin1, 2,
  • Ali Mahmoud Muddathir3Email author and
  • Tohru Mitsunaga2
BMC Complementary and Alternative MedicineBMC series – open, inclusive and trusted201717:224

DOI: 10.1186/s12906-017-1735-y

Received: 19 August 2016

Accepted: 8 April 2017

Published: 20 April 2017

Abstract

Background

Periodontal diseases are one of the major health problems and among the most important preventable global infectious diseases. Porphyromonas gingivalis is an anaerobic Gram-negative bacterium which has been strongly implicated in the etiology of periodontitis. Additionally, matrix metalloproteinases-9 (MMP-9) is an important factor contributing to periodontal tissue destruction by a variety of mechanisms. The purpose of this study was to evaluate the selected Sudanese medicinal plants against P. gingivalis bacteria and their inhibitory activities on MMP-9.

Methods

Sixty two methanolic and 50% ethanolic extracts from 24 plants species were tested for antibacterial activity against P. gingivalis using microplate dilution assay method to determine the minimum inhibitory concentration (MIC). The inhibitory activity of seven methanol extracts selected from the 62 extracts against MMP-9 was determined by Colorimetric Drug Discovery Kit. In search of bioactive lead compounds, Combretum hartmannianum bark which was found to be within the most active plant extracts was subjected to various chromatographic (medium pressure liquid chromatography, column chromatography on a Sephadex LH-20, preparative high performance liquid chromatography) and spectroscopic methods (liquid chromatography-mass spectrometry, Nuclear Magnetic Resonance (NMR)) to isolate and characterize flavogalonic acid dilactone and terchebulin as bioactive compounds.

Results

About 80% of the crude extracts provided a MIC value ≤4 mg/ml against bacteria. The extracts which revealed the highest potency were: methanolic extracts of Terminalia laxiflora (wood; MIC = 0.25 mg/ml) followed by Acacia totrtilis (bark), Ambrosia maritima (aerial part), Argemone mexicana (seed), C. hartmannianum (bark), Terminalia brownii (wood) and 50% ethanolic extract of T. brownii (bark) with MIC values of 0.5 mg/ml. T. laxiflora (wood) and C. hartmannianum (bark) which belong to combretaceae family showed an inhibitory activity over 50% at the concentration of 10 μg/ml against MMP-9. Additionally, MMP-9 was significantly inhibited by terchebulin with IC50 value of 6.7 μM.

Conclusions

To the best of our knowledge, flavogalonic acid dilactone and terchebulin were isolated from C. hartmannianium bark for the first time in this study. Because of terchebulin and some crude extracts acting on P. gingivalis bacteria and MMP-9 enzyme that would make them promising natural preference for preventing and treating periodontal diseases.

Keywords

Sudanese medicinal plants Combretum hartmannianum Porphyromonas gingivalis MMP-9 Flavogalonic acid dilacton Terchebulin

Background

Periodontal diseases are multifactorial infections caused by a specific group of Gram-negative anaerobic bacteria leading to destruction of the tooth-supporting tissue including the alveolar bone and the periodontal ligament. Two major factors contributed to the pathogenesis of periodontitis are namely periodontopathogens which cause direct damage to periodontal tissue through the secretion of toxic products, and the host response to periodontopathogens which results in the release of inflammatory mediators (proinflammatory cytokines, matrix metalloproteinases (MMPs) and prostanoids) [1].

Porphyromonas gingivalis, a Gram-negative, black pigmented and an anaerobic bacterium, has been strongly implicated in the etiology of some types of periodontitis including chronic adult periodontitis [2, 3]. It is a major periodontal pathogen that possesses multiple virulence factors including gingipains, lipopolysaccharides and can trigger host cells to release inflammatory cytokines and MMPs [4]. Previous studies showed that MMP-9 secretions were unregulated by P. gingivalis supernatant in periodontal ligament fibroblasts, pulp fibroblasts and osteosarcoma cells [57].

Most of the Sudanese people in rural areas rely on traditional medicine for the treatment of many infectious diseases (Table 1). Different plant species of medicinal importance have successfully been included in mouthwashes and toothpastes in many countries [810]. Human pathogenic microorganisms have developed resistance to drugs owing to the extensive often use of commercial synthetic antibacterial drugs in large quantities without proper medical prescriptions and tests. This condition has raised alarm in most countries and scientists are forced to search for an alternative to these compounds, often in the form of natural medicines from sources such as plants [11].
Table 1

Selected Sudanese medicinal plant species used in traditional medicine

No

Botanical names

Family

Vernacular name

Part used

Traditional uses

1

Calotropis procera (Aiton) Dryand

Apocynaceae

Ushar

Leaves

Fever, joint pains, muscular spasm, constipation, against scorpion bites, jaundice [45], healing thorn injuries [46], anti-rheumatic [47, 48].

2

Arestolochia bracteolate Lam.

Aristochiaceae

Um- Galagel

Whole plant

Malaria, HIV-1 [49, 50].

3

Xanthium brasilicum Vell.

Asteraceae

Ramtouk

Leaves

Venereal diseases, malaria [51, 52]

4

Vernonia amygdalina Delile

 

Gharib elwadi

Leaves

Fever, gastro-intestinal disease “GID” [53].

5

Adanosonia digitata L.

Bombacaceae

Tabaldi

Fruit pulp

The fruits are used as a cold beverage, added to yoghurt for treatment of diarrhea and amoebic dysentery [54].

6

Terminalia laxiflora Engl.

Combretaceae

Darut

Wood

Malaria, cough treatments, heartwood for fumigant [55, 56].

7

Terminalia brownii Fresen

 

Sobagh, Shaff

Wood, bark

Against cough and bronchitis [47], anti-rheumatic [48].

8

Combretum hartmannianum (Schweinf)

 

Habil

Wood, bark

Febrile, jaundice, bacterial infections [37, 38].

9

Ambrosia maritima L.

Compositae

Damsisa

Aerial part

The herbs are used in treatment of urinary tract infections and elimination of kidney stones, whereas the leaves are used as anti-diabetic and anti-hypertensive [57].

10

Euphorbia hirta L.

Euphorbiaceae

Um libina

Aerial part

Decoction of plant is use in asthma and bronchitis [58].

11

Ricinus communis L.

 

Khirwe

Leaves

The leaves are used as a poultice in treatment of abscesses [59, 60].

12

Acacia seyal var. fistula (Schweinf.)

Leguminosae

Sfar abide

Wood, bark

Fumigation, rheumatic pain [61].

13

Acacia seyal var. seyal Del.

 

Talih

Wood, bark

Anti-rheumatic, mouth detergent [62].

14

Acacia tortilis (Forssk.) Hayne

 

Seyyal

Wood, bark

Treat skin infection, allergic dermatomes [63].

15

Cassia acutifolia Delile

 

Senna makka

Leaves

Laxative [64], against GID [48].

16

Parkinsonia aculeata L.

 

Sesaban

Leaves

Antipyretic, anti- diabetics [63].

17

Senna italica Mill.

 

Sin elkalb

Leaves

Intestinal complications, haemomorphoids, circulatory system problems, calculi in the urinary system, sexually transmitted diseases [65].

18

Khaya senegalensis (Desv) A. Juss

Meliaceae

Mahogany

Bark

Anti-malarial, against hepatic inflammation, sinusitis, skin diseases, GID, trachoma [48].

19

Polygonum glabrum Willd

Polygonaceae

Altomsahia

Leaves

Anthelminthic, antimalarial [66].

20

Argemone mexicana L.

Papaveraceae

Argemone

Leaves, seed

Venereal diseases [52].

21

Solanum dubium Fresen

Solanaceae

Gibben

Fruits

The whole plant and fruits are pulped and applied to wounds and skin tumors as a dressing [67].

22

Salvadora persica L.

Salvadoraceae

Alarak

Leaves, stem

Gingivitis, malaria liver swellings, HIV-1 [50, 68, 69].

23

Tamarix nilotica (Ehrenb.)Bunge

Tamaricaceae

Tarft al nil

Stem

Febrile, colds [69].

24

Tribulus terrestri L.

Zygophyllaceae

Derresia

Aerial part

Demulcent, renal nephritis [47].

Combretum hartmannianum a shrub up to 4 m; as a tree under favorable conditions 10 m high. The plant is widespread throughout the Sahel belt from Senegal to Cameroon, and eastwards to the Sudan [12]. Leaves, fruits and stem bark extracts of C. hartmannianum showed activity against Gram-positive bacteria, E. coli (Gram-negative); and have also been reported to exhibit anti-inflammatory activity [13, 14].

Hence, the purpose of this study was firstly to investigate the antibacterial activity against P. gingivalis bacteria of 62 methanolic and 50% ethanolic extracts from 24 selected Sudanese medicinal plants species. Secondly, from these 62 extracts; seven methanol extracts (Terminalia laxiflora, Tamarix nilotica, Khaya senegalensis, Acacia seyal var. fistula, Acacia seyal var. seyal, C. hartmannianum and Terminalia brownii) were selected to examine their inhibitory activities against MMP-9 enzyme. Additionally methanolic extract of C. hartmannianum bark that demonstrated good combined activities were subjected for further fractionation in order to identify the active compounds responsible for the biological activities.

Methods

Plant materials

Twenty four different plant species were collected from Khartoum and Elgadarif States, Sudan, identified and authenticated by Dr. Ashraf Mohamed from the Faculty of Forestry, Mrs. Hamza Tag EL-Sir Herbarium Curator. Voucher specimens (Table 3) were deposited in the Horticultural Laboratory, Department of Horticulture, Faculty of Agriculture, University of Khartoum.

Preparation of plant extracts

Different plant parts (Table 3) were dried under shade and then grounded before they were subjected to cold maceration with methanol or 50% ethanol. The plant powder was macerated with a gentle shaking for 12 h three times in solvents in side stoppered flasks at room temperature. The extracted solvents were filtrated and evaporated under reduced pressure using a rotatory evaporator, and the concentrated 50% ethanol extracts were then dried with a freeze dryer, resulting in 62 crude extracts and stored at 4 °C until use. In order to prepare stock solution, extracts were dissolved in 100% dimethyl sulfoxide (DMSO). Further serial dilution of the stock was performed to obtain a range of desired concentration of the extracts.

Fractionation, purification and isolation of Combretum hartmannianum bark

Five grams of C. hartmannianum bark methanolic extract was subjected to fractionation by medium pressure liquid chromatography (MPLC) using ODS column (YMC-DispoPack AT ODS-25:120 g). The column was conditioned with the first eluent used for separation for 30 min with flow rate 0.5 ml/min. MPLC separation was performed by using a chromatography pump (540 Yamazen, Japan), UV detector at 280 nm wavelength (UV-10 V Yamazen, Japan) and a fraction collector (SF-2120, Advantec Tokyo Ltd., Japan). Elution with H2O/MeOH 95/5, 20/80 and absolute methanol resulted in three fractions (F1, F2 and F3). Fraction one (F1), which demonstrated a good inhibitory activity against bacteria and enzyme, was subjected to column chromatography on a Sephadex LH-20 eluted with methanol (90–20%) in water, and finally washed with 70% acetone to give five sub-fractions. Separation of these sub-fractions mainly, (F1–1, F1–2 and F1–3) were performed by using preparative high performance liquid chromatography (HPLC) with reversed phase Inertsil ODS-3 column (GL Sciences Inc. 10 mm i.d. × 250 mm) monitored at 280 nm. The solvent system used was as follows: a gradient program for 60 min from 10 to 100% methanol in water with 0.05% TFA at a flow rate 5 ml/min [15].

Compounds were identified by liquid chromatography-mass spectrometry (LC-MS) with negative ion mode and 1H,13C NMR. Methanol-d4 was used as the NMR solvent. NMR measurements were obtained by using JEOL ECP 600 MHz NMR. Spectroscopic data of flavogalonic acid dilactone and terchebulin were in good correlation to published data [16, 17] (Table 2).
Table 2

1H- and 13C–NMR data of flavogallonic acid dilactone and terchebulin (in CD3OD) as compared with literature [16, 17]

Position

Flavogallonic acid dilactone

 

Terchebulin

1H (ppm) JH,H (Hz)

1H (ppm) [16]

13C (ppm)

13C (ppm) [16]

 

1H (ppm) JH,H (Hz)

1H (ppm) [17]

13C (ppm)

13C (ppm)[17]

        

113.0

 
     

A

  

125.1

 

1

  

108.1

107.3

B

  

123.5

 
     

C

  

123.5

 
     

D

  

122.2

 
        

114.0

 
     

A

6.56 (s, H)

6.63 (s, H)

106.8

 

2

  

135.7

135.2

B

  

113.0

 
     

C

6.79 (s, H)

6.80 (s, H)

108.5

 
     

D

  

141.7

 
        

138.4

 
     

A

  

144.5

 

3

  

136.3

136.2

B

  

143.4

 
     

C

  

144.6

 
     

D

  

139.1

 
        

150.3

 
     

A

  

136.1

 

4

  

136.5

136.7

B

  

135.9

 
     

C

  

137.5

 
     

D

  

137.6

 
        

113.0

 
     

A

  

143.4

 

5

7.26 (s)

7.11(s)

112.8

111.3

B

7.48 (s, H)

7.58 (s, H)

144.5

 
     

D

  

144.6

 
     

C

  

143.6

 
        

114.0

 
     

A

  

112.0

 

6

  

110.1

109.5

B

6.37 (s, H)

6.37 (s, H)

106.4

 
     

C

  

116.0

 
     

D

6.42 (s, H)

6.39 (s, H)

106.5

 
        

159.5

160.3

7

    

A

  

168.9

169.9

   

158.9

157.1

B

  

169.5

169.1

     

C

  

167.0

167.6

     

D

  

166.9

166.8

1’

  

108.1

107.3

   

112.0

 

2’

  

137.8

137.8

   

114.0

 

3’

  

139.2

138.9

   

140.7

 

4’

  

143.2

143.1

   

147.4

 

5’

  

117.5

117.6

   

113.0

 

6’

  

114.4

109.9

   

114.0

 

7’

  

160.4

158.9

   

158.3

157.9

1”

  

124.9

125.8

 

5.23 (d, J = 2.8 Hz)

5.32 (d, J = 4 Hz)

90.2

90.8

2”

  

120.2

120.0

 

4.98 (dd, J = 3.5, 9.7 Hz)

4.88 (dd, J = 3.9 Hz)

74.2

75.1

3”

  

144.1

143.1

 

5.64 (t, J = 9.6 Hz)

5.59 (t, J = 8 Hz)

74.1

74.6

4”

  

145.9

145.8

 

4.78 (t, J = 11.0 Hz)

 

68.5

70.1

5”

  

147.8

147.5

 

4.21 (t, J = 10.3 Hz)

 

69.0

69.6

6”

7.50 (s)

7.49(s)

113.3

112.5

 

3.04 (t, J = 11.6 Hz) 4.48 (t, J = 8.9 Hz)

3.10 (d, J = 12 Hz)

63.4

64.4

7”

  

168.9

167.1

 

-

-

-

-

Determination of minimum inhibitory concentration (MIC)

MIC was determined by the broth dilution method according to Iwaki et al. [18]. Prophyromonas gingivalis ATTC 33277 was cultured in a Brain-Heart Infusion broth supplemented with 0.5 μg/ml vitamin K and 5 μg/ml hemin. The crude extracts and pure compounds were tested for antibacterial activity in sterile 96-well plates. The inoculums were prepared by diluting the broth culture to approximately 108 cell/ml. To each well; 100 μl of microbial inoculums were added and followed by addition of media to achieve a final volume of 200 μl. The tested extracts or isolated compounds were prepared in a concentration range of 4000–31.3 μg/ml using a two-fold dilution method. The experiments were performed in triplicate. Chlorhexidine was included in the assays as positive control. The cultures were incubated for 72 h at 37 °C under anaerobic conditions. Microbial growth was indicated after the addition of 50 μl of (0.2 mg/ml) p-iodonitrotetrazolium violet (INT) to the cultures and incubated at 37 °C for 2 h. The MIC was defined as the lowest concentration that inhibited the color change of INT [19].

Measurement of collagenase activity

Collagenase (MMP-9) inhibition activities of seven selected methanolic extracts and isolated compounds were investigated by using a MMP-9 Colorimetric Drug Discovery Kit: AK-404, AK-414 and AK-412 (Enzo Life Science, Plymouth, PA, USA). Briefly, aliquots (50 μl) of buffer solution were distributed into a 96 well plate. Twenty microliter of each diluted MMP-9, methanolic extracts or isolated compounds at different concentrations were added and reaction mixtures were incubated for 30 min at 37 °C and diluted substrate (thiopeptide; 10 μl) was added. N-Isobutyl-N-(4-methoxyphenylsulfonyl) glycyl hydroxamic acid (NNGH) was used as positive control. Inhibition was measured by continuously reading plates at absorbance 414 nm for 10 min in a microplate reader. All assays were performed independently in triplicate [20]. The inhibition of MMP-9 was calculated using the formula:
$$ \mathrm{Inhibition}\%=\left[100\hbox{-} \left(\mathrm{VI}/\mathrm{VC}\right)\right]\times 100 $$

Where:

VI: reaction velocity of (sample or inhibitor).

VC: reaction velocity of control.

Statistical analysis

The percentage and IC50 values of MMP-9 inhibitory activities were expressed as the mean value. The significant differences between extracts or isolated compounds were assessed by one-way analysis of variance (ANOVA) followed by pair wise comparison of the means using Tukey’s multiple comparison test. Values were determined to be significant when p was less than 0.05 (p < 0.05).

Results and discussion

In this study methanol and 50% ethanol were chosen as solvent for extraction. As shown in the previous studies, nearly all of the identified components from plants active against microorganisms and enzyme may be related to the polyphenolic content of the plant extract, so the initial screenings of plants can be done by using crude aqueous or alcohol extraction [21, 22].

Evaluation of MIC activity of plant extracts against P.gingivalis

In our search for natural products with beneficial properties for oral health, we evaluated the ability of 24 Sudanese medicinal plants species belonging to 15 families in order to reveal the inhibitory activity against P.gingivalis bacteria. Botanical name, part used, voucher specimen and MIC activity of methanol and 50% ethanol extracts against P. gingivalis were shown in Table 3. Comparatively, methanol extracts displayed better anti-P. gingivalis activity than 50% ethanol extracts. Among 62 plant extracts; 50 extracts exhibited MIC activity at the concentration of 4 mg/ml or less; moderate inhibitory activity (MIC = 1 mg/ml) were found in sixteen plant extracts. The most potent extracts were methanol extract of Terminalia laxiflora (MIC value 0.25 mg/ml) followed by Ambrosia maritima, Argemone mexicana (seed), Terminalia brownii (wood), C. hartmannianum (bark), Acacia totrtilis (bark) and 50% ethanolic extract of T. brownii (bark) with MIC value 0.5 mg/ml (Table 3). According to the reported previous studies, T. laxiflora also showed potent antibacterial activity against Propionibacterium acne with MIC value 0.13 mg/ml and their activity was due to hydrolizable tannins [23].
Table 3

Minimum inhibitory concentration (MIC) activities of selected Sudanese medicinal plants against P. gingivalis

Botanicals name

Examined part

Voucher specimen

MIC mg/ml

MeOH

50% EtOH

A. bracteolate Lam.

Whole plant

SD-SH-04

2

1

A. digitata L.

Fruit pulp

SD-OD-27

2

4

A. maritima L.

Aerial part

SD-SH-03

0.5

2

A. mexicana L.

Leaves

SD-KH-39

-

- a

 

Seed

 

0.5

2

A. tortilis (Forssk.) Hayne

Bark

SD-KH-07

0.5

4

 

Wood

 

-

4

A.seyal var. fistula (Schweinf.)

Bark

SD-GF-06

1

4

 

Wood

 

-

-

A.seyal var. seyal Del.

Bark

SD-GF-05

1

2

 

Wood

 

-

-

C. hartmannianum (Schweinf)

Bark

SD-KH-04

0.5

1

 

Wood

 

1

-

C. procera (Aiton) Dryand

Leaves

SD-SH-11

2

4

C.acutifolia Delile

Leaves

SD-SH-24

1

1

E. hirta L.

Aerial part

SD-SH-37

2

4

K. senegalensis (Desv) A. Juss

Bark

SD-SH-14

1

2

P. aculeata L.

Leaves

SD-SH-02

1

4

P. glabrum Willd

Leaves

SD-SH-A-03

1

-

R. communis L.

Leaves

SD-SH-36

1

4

S. dubium Fresen

Fruits

SD-SH-34

-

2

S. italica Mill.

Leaves

SD-SH-25

2

2

S. persica L.

Stem

SD-SH-09

1

4

 

Leaves

 

-

-

T. brownii Fresen

Bark

SD-GF-02

1

0.5

 

Wood

 

0.5

2

T. laxiflora Engl.

Wood

 

0.25

2

T. nilotica (Ehrenb.)Bunge

Stem

SD-OD-10

2

4

T. terrestri L.

Aerial part

SD-SH-33

1

1

V. amygdalina Delile

Leaves

SD-KH-19

1

2

X. brasilicum Vell.

Leaves

SD-SH-12

2

2

a:has no activity up to 4 mg/ml, MeoH: Methanol, 50% EtOH: 50% Ethanol

chlorohexidine as positive control has MIC value 0.0004 mg/ml

Also noteworthy the combrataceae family; C. hartmannianum (bark), T. brownii (wood and bark) and T. laxiflora demonstrated inhibitory activity against P. gingivalis with MIC values 2 mg/ml or less, except 50% ethanol extract of C. hartmannianum (wood) had no activity up to 4 mg/ml. This family has a wide range of tannins, flavonoids, terpenoids and stilbenoids [24, 25]. Flavonoids have been reported to be mainly active against Gram-negative bacteria [26]. In this study, methanolic extract of C. hartmannianum (bark) exhibited good activity against P. gingivalis (MIC 0.5 mg/ml), and this was in agreement with Eldeen and Van [27] who reported that bark of C. hartmannianum inhibited the growth of Gram-negative bacteria at a concentration less than/or around 1.56 mg/ml.

The positive control (chlorhexidine) showed a significant inhibitory activity compared to the other extracts. However, chlorhexidine has several side effects such as undesirable tooth discoloration, unpleasant taste and causing dryness and burning sensation in the mouth, leading to patient dissatisfaction [28, 29].

Inhibitory activities of selected methanolic plants extracts against MMP-9

Several therapeutic strategies, based on targeting different pathways of the pathogenesis of periodontal disease, have been put forward. In this regard, a number of authors proposed that periodontitis progression could be hampered by successfully inhibiting both bacteria and host-derived proteinases involved in connective tissue destruction of the periodontium [30, 31].

From our previous study to explore a natural agent for preventing and treatment of dental cavity, seven Sudanese methanolic extracts namely; T. laxiflora (wood), Tamarix nilotica (stem) and bark of Khaya senegalensis‚ Acacia seyal var. fistula, Acacia seyal var. seyal, C. hartmannianum and T. brownii showed potent inhibitory activity against glucosyltransferase enzyme that promotes the binding of cariogenic bacteria on the teeth (Additional file 1: Table S1). Therefore these seven methanolic extracts were selected for assayed their ability to inhibit the MMP-9 enzyme.

All seven selected extracts exhibited activity higher than 50% inhibition at the concentration of 100 μg/ml against MMP-9 (Fig. 1). NNGH (positive control) recorded 100% inhibition at the concentration of 100 μg/ml. At the concentration of 100 μg/ml, methanolic extracts of T. laxiflora and T. brownii (bark) significantly inhibited MMP-9. Considerable, but less potent methanolic bark extracts of A. seyal var. seyal, C. hartmannianum and A. seyal var. fistula exhibited MMP-9 inhibitory activity at the concentration of 100 μg/ml. Nevertheless, at the concentration 10 μg/ml, T. laxiflora showed the potent inhibitory activity against MMP-9 followed by C. hartmannianum (bark). Kusumoto et al. [32] mentioned that the stem bark of Terminalia arjuna inhibited the HIV-1 protease activity by more than 70% at a concentration of 0.2 mg/mL. Pomegranate methanol extract inhibited the secretion of MMP-9; this inhibitory effect was likely to be due to hydrolysable tannins [33]. Hydrolyzable tannins were suggested to exhibit their inhibitory effect on the tumor cell invasion via direct inhibition of MMP-9 activity [34]. Seigler [35] stated that Acacia spp. contained hydrolyzable tannins, flavonoids and condensed tannins.
Fig. 1

Inhibitory activities of seven methanolic plants extracts against MMP-9. B: bark. NNGH as positive control has 100% inhibition at concentration 100 μg/ml. Values were expressed as mean ± SD, n = 3. Values not followed by a common letter were significantly different at the level (p < 0.05)

Inhibitory activities of compounds isolated from C. hartmannianium bark against P. gingivalis and MMP-9

In the present study, some plants belong to combretaceae family revealed good inhibitory activities against P. gingivalis and MMP-9; such as methanolic extracts of T. laxiflora, C. hartmannianium (bark) and T. brownii (bark). The potency of T. laxiflora was probably due to the presence of terchebulin and flavogalonic acid dilactone in wood at high concentration [15]. Kosei et al. [36] isolated gallic acid, punicalagin, terchebulin, ellagic acid 4-O-α-L-rhamnopyranoside, ellagic acid, and 3, 4, 3’-tri-O-methylellagic acid from methanolic extracts of T. brownii bark. However, there is no data was reported in literature regarding the isolated compounds from C. hartmannianium species bark, which makes it a potential candidate for further separation and isolation of compounds.

Among antimicrobial active compounds isolated from Combretum spp. are; combretastatins, acidic tetracyclic and pentacyclic triterpenes/triterpenoids, ellagitannins, phenanthrenes, flavonoids and saponins [37, 38]. C. hartmannianum gave good activity against Gram-positive and Gram-negative bacteria, and the most of the activity was found in water and methanol extracts. Additionally, the extracts of C. hartmannianum were found to be active against enzymes such as reverse transcriptase and tyrosine kinase [39].

Bioassay guided fractionation led to the isolation of two compounds namely, flavogalonic acid dilactone and terchebulin (Figs. 2, 3). Terchebulin, and to a lesser extent flavogalonic acid dilactone showed combined activity against P. gingivalis and MMP-9 (Table 4). Marquis et al. [40] reported that polyphenols reduced MMP-9 activity and P. gingivalis growth; since polyphenols were reported to possess antimicrobial and anti-inflammatory properties, they might be of interest as therapeutic agents for controlling periodontal diseases, which involved both pathogenic bacteria and host immune responses.
Fig. 2

Flavogallonic acid dilactone. Tan powder. LC-MS (negative ion mode) m/z: 469 (M-H); 1H–NMR (in CD3OD): δ (ppm) 7.26 (s), 7.50 (s). 13C–NMR (in CD3OD): δ (ppm) 108.1 (C-1, 1′), 110.1–114.4 (C-6, 6′), 112.8 (C-5), 113.3 (C-6″), 117.5–120.2 (C-5″), 124.9 (C-1″), 135.7 (C-2), 136.3 (C-3), 136.5 (C-4), 137.8 (C-2′), 139.2 (C-3′), 143.2 (C-4′), 144.1 (C-3″), 145.9 (C-4″), 147.8 (C-5″), 158.9–160.4 (C-7, 7′), 168.9 (C-7″)

Fig. 3

Terchebulin. Tan powder. LC-MS (negative ion mode) m/z: 1083 (M-H); 1H–NMR (in CD3OD): δ (ppm) 3.04 (t, J = 11.6 Hz, one of the H-6″), 4.21 (t, J = 10.3 Hz, H-5″), 4.48 (t, J = 8.9 Hz, one of the H-6″), 4.78 (t, J = 11.0 Hz, H-4″), 4.98 (dd, J = 3.5, 9.7 Hz, H-2″), 5.23 (d, J = 2.8 Hz, H-1″), 5.64 (t, J = 9.6 Hz, H-3″), 6.37 (s, H-B6), 6.42 (s, H-D6), 6.56 (s, H-A2), 6.79 (s, H-C2), 7.48 (s, H-5). 13C–NMR (in CD3OD): δ (ppm) 63.4 (C-6″), 68.5 (C-4″), 69.0 (C-5″), 74.1 (C-3″), 74.2 (C-2″), 90.2 (C-1″), 106.4 (C-B6), 106.5 (C-D6), 106.8 (C-A2), 108.5(C-C2), 112.0–114.0 (C-A6, B2, 5, 5′, 1, 1′, 2, 2′, 6, 6′), 116.0 (C-C6), 122.2 (C-D1), 123.5 (C- B1,C1), 125.1 (C-A1), 135.9 (C-B4), 136.1 (C-A4), 137.5 (C-C4), 137.6 (C-D4), 138.4 (C-3), 139.1 (C-D3), 140.7 (C-3′), 141.7 (C-D2), 143.4–143.6 (C-A5, B3, C5), 144.5–144.6 (C-A3, B5, C3, D5), 147.4 (C-4′), 150.3 (C-4), 158.3 (C-7′), 159.5 (C-7), 166.9 (C-D7), 167.0 (C-C7), 168.9 (C-A7), 169.5 (C-B7)

Table 4

Minimum inhibitory concentration (MIC) and matrix metalloproteinases −9 (MMP-9) inhibitory activities of isolated compounds from Combretum hartmannianium bark

Compounds

MIC (μg/ml)

*IC50 against MMP-9 (μM)

Terchebulin

500

6.7 ± 1.5a

Flavogallonic acid dilacton

1000

36.1 ± 7.5b

*IC50-Half minimal inhibitory concentration

Means with different letters in the same column were significantly different at the level (p < 0.05); n = 3

Terchebulin and flavogalonic acid dilactone had moderate antibacterial activity with MIC values of 500 and 1000 μg/ml, respectively. Previous studies showed that flavogalonic acid dilactone, terchebulin and punicalagin isolated from Terminalia spp. demonstrated antibacterial activity against P. acnes and Helicobacter pylori in a range between 125 to 250 μg/ml [15, 41]. Terchebulin demonstrated more potent activity (6.7 μM) than flavogalonic acid dilactone against MMP-9. Moreover, terchebulin has more reliable activity than chlorhexidine that inhibits MMP-9 at the IC50 25.2 μM [42]. Furthermore, Arabaci et al. [43] found that chlorhexidine had a few genotoxic and cytotoxic effects on human lymphocytes. Studies of the in vitro cytotoxic activity on mouse fibroblasts of terchebulin and flavogalonic acid dilactone showed activity at minimum cytotoxic concentration of ≥1500 μg/ml (1348, 3192 μM respectively) [44]. To the best of our knowledge, hydrolysable tannins mainly, terchebulin and flavogalonic acid dilactone were isolated from C. hartmannianium bark for the first time during this study.

Conclusions

Our study demonstrated that some methanolic crude extracts of Sudanese medicinal plants possessed good combined activities against P. gingivalis and MMP-9. Moreover, this study provided new information on terchebulin and flavogalonic acid dilactone which were isolated from methanolic extracts of C. hartmannianium bark, indicating that they possessed interesting inhibitory properties against P. gingivalis and MMP-9, and this may be useful for the prevention and treatment of periodontal diseases. Further studies are recommended to investigate the mechanisms of action of these isolated compounds, toxicity and their usefulness as a source of new components in mouthwashes and toothpastes.

Declarations

Acknowledgements

The authors would like to thank prof. Abdelkhalig Muddathir and Dr. Farouk Hassan Eltahir for the English editing of the manuscript.

Funding

No fund was available.

Availability of data and materials

Not applicable.

Authors’ contribution

E.A.M.M participated in the design of the study, antibacterial, enzyme assay, isolation of compounds and write the manuscript. A.M participated in collection, extraction of plant samples and helped to draft the manuscript. T.M supervised and designs this study. All the authors read and approved the final version of the manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Faculty of Pharmacy, University of Science and Technology
(2)
Department of Applied Biological Science, Faculty of Applied Biological Science, Gifu University
(3)
Department of Horticulture, Faculty of Agriculture, University of Khartoum

References

  1. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent Jr RL. Microbial complexes in subgingival plaque. J Clin Periodontol. 1998;25:134–44.View ArticlePubMedGoogle Scholar
  2. Slots J, Bragd L, Wikstro MM, Dahlen G. The occurrence of actinobacillus actinomycetemcomitans, bacteroides gingivalis bacteroides intermedius in destructive periodontal disease in adults. J Clin periodontal. 1986;13:570–7.View ArticleGoogle Scholar
  3. Holt SC, Felton J, Brunsvold M, Kornman KS. Implantation of bacteroide gingivalis in non-human primate initiates progression of periodontitis. Science (WashDC). 1988;1239:55–7.View ArticleGoogle Scholar
  4. Schwartz Z, Goultschin J, Dean DD, Boyan BD. Mechanisms of alveolar bone destruction in periodontitis. Periodontol. 1997;14:158–72.View ArticleGoogle Scholar
  5. Chang YC, Lai CC, Yang SF, Chan Y, Hsieh YS. Stimulation of matrix metalloproteinases by black-pigmented bacteroides in human pulp and periodontal ligament cell cultures. J Endodon. 2002;28:90–3.View ArticleGoogle Scholar
  6. Chang YC, Chu SC, Yang SF, Hsieh YS, Yang LC, Huang FM. Examination of the signal transduction pathways leading to activation of gelatinolytic activity by interleukin-1 and Porphyromonas gingivalis in human osteosarcoma cells. J Periodontal Res. 2004;39:168–74.View ArticlePubMedGoogle Scholar
  7. Chang YC, Yang SF, Lai CC, Liu JY, Hsieh YS. Regulation of matrix metalloproteinase production by cytokines, pharmacological agents and periodontal pathogens in human periodontal ligament fibroblast cultures. J Periodontal Res. 2002;37:196–203.View ArticlePubMedGoogle Scholar
  8. Varghese J, Tumkur VK, Ballal V, Bhat GS. Antimicrobial effect of Anacardium occidentale leaf extract against pathogens causing periodontal disease. Adv Biosci Biotechnol. 2013;4:15–8.View ArticleGoogle Scholar
  9. Ohara A, Saito F, Matsuhisa T. Screening of antibacterial activities of edible plants against Streptococcus mutans. Food Sci Technol Res. 2008;14:190–3.View ArticleGoogle Scholar
  10. Palombo EA. Traditional plant extracts and natural products with activity against oral bacteria: potential application in the prevention and treatment of oral diseases. Evid Based Complement Alternat Med. 2011;201:1–15.View ArticleGoogle Scholar
  11. Patra JK, Kim ES, Oh K, Kim HJ, Kim Y, Baek KH. Antibacterial effect of crude extract and metabolites of Phytolacca americana on pathogens responsible for periodontal inflammatory diseases and dental caries. BMC Complement Altern Med. 2014;14:343.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Maydell HJV. Trees and Shrubs of the Sahel. Their Characteristics and Uses, (GTZ) GmbH, Germany, 1990.
  13. Elegami AA, El-Nima EI, El Tohami MS, Muddathir AK. Antimicrobial activity of some species of the family Combretaceae. Phytother Res. 2002;16:555–61.View ArticlePubMedGoogle Scholar
  14. Eldeen MS, Van Staden J. Anti-inflammatory and mycobacterial activity of leaf extracts of Coleonema album. South Afr J Bot. 2008;74:345–7.View ArticleGoogle Scholar
  15. Muddathir AM, Mitsunaga T, Kosei Y. Anti-acne activity of tannin-related compounds isolated from Terminalia laxiflora. J Wood Sci. 2013;5:426–31.View ArticleGoogle Scholar
  16. Hirano Y, Kondo R, Sakai K. 5α-Reductase inhibitory tannin-related compound isolated from Shorea laevifolia. J Wood Sci. 2003;49:339–42.View ArticleGoogle Scholar
  17. Lin TC, Nonaka GI, Nishioka I, Ho FC. Tannins and related compounds, CII. Structures of terchebulin, an ellagitannin having a novel tetraphenyl carboxylic acid (terchebulic acid) moiety, and biogenetically related tannins from Terminalia chebula Retz. L. Chem Pharm Bull. 1990;38:3004–8.View ArticleGoogle Scholar
  18. Iwaki K, Koya-Miyata S, Kohno K, Ushio S, Fukuda S. Antimicrobial activity of Polygonum tinctorium lour: extract against oral pathogenic bacteria. J Nat Med. 2006;60:121–5.View ArticleGoogle Scholar
  19. Eloff JN. Antibacterial activity of Marula (Sclerocarya birrea (a. Rich) Hochst. Subsp. Caffra (Sond) Kokwaro) (Anacardiaceae) bark and leaves. J Ethnopharmacol. 2001;76:305–8.View ArticlePubMedGoogle Scholar
  20. Choi JS1, Ha YM, Joo CU, Cho KK, Kim SJ, Choi IS. Inhibition of oral pathogens and collagenase activity by seaweed extracts. J Environ Biol. 2012;33:115–21.PubMedGoogle Scholar
  21. Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev. 1999;12:564–82.PubMedPubMed CentralGoogle Scholar
  22. Gulati V, Harding IH, Palombo EA. Enzyme inhibitory and antioxidant activities of traditional medicinal plants: potential application in the management of hyperglycemia. BMC Complement Altern Med. 2012;12:77.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Muddathir AM, Mitsunaga T. Evaluation of anti-acne activity of selected Sudanese medicinal plants. J Wood Sci. 2013;59:73–9.View ArticleGoogle Scholar
  24. Eloff JN, Katerel DR, McGaw LJ. The biological activity and chemistry of the southern African Combretaceae. J Ethnopharmacol. 2008;119:686–99.View ArticlePubMedGoogle Scholar
  25. Martini ND, Katerere DRP, Eloff JN. Biological activity of seven antibacterial flavonoids isolated from Combretum erythrophyllum (Combretaceae). J Ethnopharmacol. 2004;93:207–12.View ArticlePubMedGoogle Scholar
  26. Basile A, Giordano S, Lopez-Saez JA, Cobianchi RC. Antibacterial activity of pure flavonoids isolated from mosses. Phytochemistry. 1999;52:1479–82.View ArticlePubMedGoogle Scholar
  27. Eldeen IMS, Van SJ. In vitro pharmacological investigation of extracts from some trees used in Sudanese traditional medicine. South Afr J Bot. 2007;73:435–40.View ArticleGoogle Scholar
  28. Zanatta FB, Antoniazzi RP, Rösing CK. Staining and calculus formation after 0.12% chlorhexidine rinses in plaque-free and plaque covered surfaces: a randomized trial. J Appl Oral Sci. 2010;18:515–21.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Amanlou M, Beitollahi JM, Abdollahzadeh S, Tohidast-Ekrad Z. Miconazole gel compared with Zataria multiflora Boiss. Gel in the treatment of denture stomatitis. Phytother Res. 2006;20:966–9.View ArticlePubMedGoogle Scholar
  30. Kitano S, Irimura K, Sasaki T, Abe N, Baba A, Miyake Y, Katunuma N, Yamamoto K. Suppression of gingival inflammation induced by Porphyromonas gingivalis in rats by leupeptin. Jpn J Pharmacol. 2001;85:84–91.View ArticlePubMedGoogle Scholar
  31. Preshaw PM, Hefti AF, Jepsen S, Etienne D, Walker C, Bradshaw MH. Subantimicrobial dose doxycycline as adjunctive treatment for periodontitis. A review. J Clin Periodontol. 2004;31:697–707.View ArticlePubMedGoogle Scholar
  32. Kusumoto T, Nakabayashi T, Kida H, Miyashiro H, Hattori M, Namba T. Screening of various plant extracts used in Ayurvedic medicine for inhibitory effects on human immunodeficiency virus type 1 (HIV-1) protease. Phytother Res. 1995;9:180–4.View ArticleGoogle Scholar
  33. Dell'Agli M, Galli GV, Bulgari M, Basilico N, Romeo S, Bhattacharya D, Taramelli D, Bosisio E. Ellagitannins of the fruit rind of pomegranate (Punica granatum) antagonize in vitro the host inflammatory response mechanisms involved in the onset of malaria. Malar J. 2010;9:208.View ArticlePubMedPubMed CentralGoogle Scholar
  34. Folgueras AR, Pendas AM, Sanchez LM, Lopez-Otin C. Matrix metalloproteinases in cancer: from new functions to improved inhibition strategies. Int J Dev Biol. 2004;48:411–24.View ArticlePubMedGoogle Scholar
  35. Seigler DS. Phytochemistry of Acacia-sensu lato. Biochem Syst Ecology. 2003;31:845–73.View ArticleGoogle Scholar
  36. Kosei Y, Mitsunaga T, Ali MM. Screening for melanogenesis-controlled agents using Sudanese medicinal plants and identification of active compounds in the methanol extract of Terminalia brownii bark. J Wood Sci. 2016;62:285–93.View ArticleGoogle Scholar
  37. El Ghazali GEB, El Tohamy MS, El Egami AB. Medicinal plants of the Sudan. Part III. Sudan: Medicinal Plants of the White Nile Province Khartoum University Press; 1994.Google Scholar
  38. Al Magboul AZ, Bashir AK, Salih AM, Farouk A, Khalid SA. Antimicrobial activity of certain Sudanese plants used in folkloric medicine: screening for antibacterial activity (V). Fitoterapia. 1988;59:57–62.Google Scholar
  39. Ali H, König GM, Khalid SA, Wright AD, Kaminsky R. Evaluation of selected Sudanese medicinal plants for their in vitro activity against hemoflagellates, selected bacteria, HIV-1-RT and tyrosine kinase inhibitory, and for cytotoxicity. J Ethnopharmacol. 2002;83:219–28.View ArticlePubMedGoogle Scholar
  40. Marquis A, Genovese S, Epifano F, Grenier D. The plant coumarins auraptene and lacinartin as potential multifunctional therapeutic agents for treating periodontal disease. BMC Complement Altern Med. 2012;2:80.Google Scholar
  41. Silva O, Viegas S, de Mello-Sampayo C, Costa MJ, Serrano R, Cabrita J, Gomes ET. Anti-Helicobacter pylori activity of Terminalia macroptera root. Fitoterapia. 2012;83:872–6.View ArticlePubMedGoogle Scholar
  42. Ohtsuki T, Yokosawa E, Koyano T, Preeprame S, Kowithayakorn T, Sakai S, Toida T, Ishibashi M. Quinic acid esters from Pluchea indica with collagenase, MMP-2 and MMP-9 inhibitory activities. Phytother Res. 2008;22:264–6.View ArticlePubMedGoogle Scholar
  43. Arabaci T, Türkez H, Çanakçi CF, Özgöz M. Assessment of cytogenetic and cytotoxic effects of chlorhexidine digluconate on cultured human lymphocytes. Acta Odontol Scand. 2013;71:1255–60.View ArticlePubMedGoogle Scholar
  44. Shuaibu MN, Wuyep PA, Yanagi T, Hirayama K, Tanaka T, Kouno I. The use of microfluorometric method for activity-guided isolation of antiplasmodial compound from plant extracts. Parasitol Res. 2008;102:1119–27.View ArticlePubMedGoogle Scholar
  45. Mossa JS, Tarig M, Mohsin A, Rafatulla S. Pharmacological studies on aerial parts of Calotropis procera. Am J Chin Med. 1991;19:223–31.View ArticlePubMedGoogle Scholar
  46. El Ghazali GEB, Bari EA, Bashir AK, Salih AM. Medicinal plants of Sudan part II. Medicinal plants of eastern Nuba Mountains. Khartoum: National Council for Research; 1987.Google Scholar
  47. El Ghazali GEB, El Tohami MS, El Egami AA, Abdalla WE, Galal M. Medicinal plants of the Sudan part IV. Medicinal plants of north Kordofan. Khartoum: National Council for Research; 1997.Google Scholar
  48. El Ghazali GEB, Abdalla WE, Khalid HE, Khalafalla MM, Hamad AA. Medicinal plants of the Sudan part V. Medicinal plants of Ingassana area. Khartoum: National Council for Research; 2003.Google Scholar
  49. El Kamali HM, El Khalifa KF. Folk medicinal plants of riverside forests of the southern Blue Nile district. Sudan Fitoterapia. 1999;70:493–7.View ArticleGoogle Scholar
  50. Hussein G, Miyashiro H, Nakamura N, Hattori M, Kawahata T, Otake T. Inhibitory effects of Sudanese plant extracts on HIV-1 replication and HIV-1 protease. Phytother Res. 1999;13:31–6.View ArticlePubMedGoogle Scholar
  51. Nour AM, Khalid SA, Kaiser M, Brun R, Abdallah WE, Schmidt TJ. The antiprotozoal activity of sixteen asteraceae species native to Sudan and bioactivity-guided isolation of xanthanolides from Xanthium brasilicum. Planta Med. 2009;75:1363–8.View ArticlePubMedGoogle Scholar
  52. Dahab MM, Koko WS, Osman EE. In vitro antitrichomonal activity of Xanthium brasilicum vell and Argemone mexicana L different extracts. J Med Plant Res. 2011;5:151–5.Google Scholar
  53. Rahman MA, Mossa JS, AL-Said MS, AL-Yahya MA. Medicinal plant diversity in the flora of Saudi Arabia 1: a report on seven plant families. Fitoterapia. 2004;75:149–61.View ArticlePubMedGoogle Scholar
  54. Kinghorn AD, Chai HB, Sung CK, Keller WJ. The classical discovery approach to defining bioactive constituent of botanicals. Fitoterapia. 2011;82:71–9.View ArticlePubMedGoogle Scholar
  55. Musa S, Abdelrasool F, El E, Ahmed L, Mahmoud A, Yagi S. Ethnobotanical study of medicinal plants in the Blue Nile state, South-eastern Sudan. J Med Plants Res. 2011;5:4287–97.Google Scholar
  56. Ogbazghi W, Bein E. Assessment of non-wood forest products and their role in the livelihoods of rural communities in the gash-Barka region, Eritrea. Drylands Coordination Group Report. 2006;40:26–7.Google Scholar
  57. Mahmoud AA, Ahmed AA, El Bassuony AA. A new chlorosesquiterpene lactone from Ambrosia maritima. Fitoterapia. 1999;70:575–8.View ArticleGoogle Scholar
  58. Hatil HE. Medicinal plants in east and central Africa: challenges and constraints. Ethnobot leaflet. 2009;13:364–9.Google Scholar
  59. Ferraz AC, Angelucci ME, Da Costa ML, Batista IR, De Oliveira BH, Da Cunha C. Pharmacological evaluation of ricinine, a central nervous system stimulant isolated from Ricinus communis. Pharmacol Biochem Behav. 1999;63:367–75.View ArticlePubMedGoogle Scholar
  60. Holfelder MG, Steck M, Komor E, Seifert K. Ricinine in phloem sap of Ricinus communis. Phytochemistry. 1998;47:1461–3.View ArticleGoogle Scholar
  61. Duke JA. Medicinal plants of the bible. Owerri, NY: Trado-Medic Books; 1983.Google Scholar
  62. Mangan JL. Nutritional effects of tannins on animal feeds. Nutr Res Rev. 1988;1:209–31.View ArticlePubMedGoogle Scholar
  63. Louhaichi M, Salkini AK, Estita HE, Belkhir S. Initial assessment of medicinal plants across the Libyan Mediterranean coast. Adv Environ Biol. 2011;5:359–70.Google Scholar
  64. El Ghazali GEB. Medicinal plants of Sudan part I. Khartoum: Medicinal Plants of Erkawit, National Council for Research; 1986.Google Scholar
  65. Masoko P, Gololo SS, Mokgotho MP, Eloff JN, Howard RL, Mampuru LJ. Evaluation of the antioxidant, antibacterial, and antiproliferative activities of the acetone extract of the roots of Senna italica (Fabaceae). Afr J Tradit Complement Altern Med. 2010;7:138–48.Google Scholar
  66. Hatil HE. Effect of certain medicinal plants extracts against storage pest, Tribolium castaneum Herbst. Am Eurasian J Sustain Agric. 2009;3:139–42.Google Scholar
  67. El Kheir YM, Salih MH. Investigation of certain plants used in Sudanese folk medicine. Fitoterapia. 1980;51:143–7.Google Scholar
  68. El Kamali HM, Khalid SA. The most common herbal remedies in Central Sudan. Fitoterapia. 1996;57:301–6.Google Scholar
  69. Neuwinger DH. African traditional medicine. A dictionary of plant use and application. Stuttgart, Germany: Med. Pharm. Pub; 2000.Google Scholar

Copyright

© The Author(s). 2017

Advertisement